The discovery of Rous sarcoma virus (RSV) led to the identification of a cellular oncogene Src (c-Src), which encodes a non-receptor tyrosine kinase (phosphoprotein of molecular weight 60,000 Dalton or pp60 c-Src). The Src oncogene has been implicated in the development of numerous types of cancers via a yet to be elucidated mechanism (see for example Stehelin, D., Varmus, H. E., Bishop, J. M. & Vogt, P. K. Nature 260, 170-173 (1976); Brugge, J. S. & Erikson, R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature 269, 346-348 (1977); Jove, R. & Hanafusa, H. Cell transformation by the viral Src oncogene. Annu Rev Cell Biol 3, 31-56 (1987); Thomas, S. M. & Brugge, J. S. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13, 513-609 (1997)). The nucleic acid sequence of normal c-Src is as follows:
atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac ggagggagac360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat caaaaccctg900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag1560 gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g1611
The c-Src nucleic acid sequence encodes for a tyrosine kinase protein pp60, which has a following sequence:
  1 MGSNKSKPKD ASQRRRSLEP AENVHGAGGG AFPASQTPSK PASADGHRGP SAAFAPAAAE  61 PKLFGGFNSS DTVTSPQRAG PLAGGVTTFV ALYDYESRTE TDLSFKKGER LQIVNNTEGD 121 WWLAHSLSTG QTGYIPSNYV APSDSIQAEE WYFGKITRRE SERLLLNAEN PRGTFLVRES 181 ETTKGAYCLS VSDFDNAKGL NVKHYKIRKL DSGGFYITSR TQFNSLQQLV AYYSKHADGL 241 CHRLTTVCPT SKPQTQGLAK DAWEIPRESL RLEVKLGQGC FGEVWMGTWN GTTRVAIKTL 301 KPGTMSPEAF LQEAQVMKKL RHEKLVQLYA VVSEEPIYIV TEYMSKGSLL DFLKGETGKY 361 LRLPQLVDMA AQIASGMAYV ERMNYVHRDL RAANILVGEN LVCKVADFGL ARLIEDNEYT 421 ARQGAKFPIK WTAPEAALYG RFTIKSDVWS FGILLTELTT KGRVPYPGMV NREVLDQVER 481 GYRMPCPPEC PESLHDLMCQ CWRKEPEERP TFEYLQAFLE DYFTSTEPQY 531 QPGENL
Amino acids are abbreviated as I-letter codes and corresponding 3-letter codes as follows: Alanine is A or Ala; Arginine R or Arg, Asparagine N or Asn; Aspartic acid D or Asp; Cysteine C or Cys; Glutamine Q or Gln; Glutamic acid E or Glu; Glycine G or Gly; Histidine H or His; Isoleucine I or Ile; Leucine L or Leu; Lysine K or Lys; Methionine M or Met; Phenylalanine F or Phe; Proline P or Pro; Serine S or Ser; Threonine T or Thr; Tryptophan W or Trp; Tyrosine Y or Tyr; and Valine V or Val.
The cellular Src oncogene (c-Src) is the normal counterpart of the transforming viral Rous sarcoma oncogene (v-Src). v-Src has been shown to induce the production of specific metalloproteinases (Hamaguchi, M. et al. Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of Src. Oncogene 10, 1037-1043 (1995)) and to foster the metastatic phenotype (Egan, S. et al. Transformation by oncogenes encoding protein kinases induces the metastatic phenotype. Science 238, 202-205 (1987); Tatsuka, M. et al. Different metastatic potentials of ras- and Src-transformed BALB/c 3T3 A31 variant cells. Mol. Carcinog. 15, 300-308 (1996)). However, as opposed to cellular c-Src the retroviral v-Src has 19 C-terminal residues replaced by a sequence of 12 amino acids, lacking the regulatory tyrosine.
The non receptor tyrosine kinase c-Src consists of an SH3, SH2 and tyrosine kinase domain. c-Src appears to be the most important to the normal function of osteoclasts, as determined from studies of Src-knock-out mice (see for example U.S. Pat. No. 5,541,109). The catalytic activity of c-Src and other nonreceptor tyrosine kinases is inhibited by the intramolecular association of their intrinsic SH2 domain to the carboxy-terminal tail upon phosphorylation of Tyr (position 530, avian position 527). Protein tyrosine phosphorylation is believed to be an important regulatory event in cell growth and differentiation. Phosphorylation on tyrosine can either decrease or increase the enzymatic activity of substrate proteins. Tyrosine phosphorylated sequences associate with Src homology 2 (SH2) domains, and thus tyrosine phosphorylation also serves to regulate protein/protein interactions. Many protein tyrosine kinases have been described to date: several are the receptors for peptide growth factors; others are expressed in the cytoplasm and nucleus. Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). There are 19 known families of receptor tyrosine kinases including the Her family (EGFR, Her 2, Her 3, Her 4), the insulin receptor family (insulin receptor, IGF-1R, insulin-related receptor), the PDGF receptor family (PDGF-R alpha and beta, CSF-1R, Kit, Flk2), the Flk family (Flk-1, Flt-1, Flk-4), the FGF-receptor family (FGF-Rs 1 through 4), the Met family (Met, Ron), etc. There are 11 known families of non-receptor type tyrosine kinases including the Src family (Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, Yrk), Abl family (Abl, Arg), Zap 70 family (Zap 70, Syk) and Jak family (Jak 1, Jak 2, Tyk 2, Jak 3). Many of these tyrosine kinases have been found to be involved in cellular signaling pathways leading to pathogenic conditions such as cancer, psoriasis, hyperimmune response, etc. Other roles for tyrosine kinases include cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiating developmental processes in both vertebrates and invertebrates.
Among various types of tumors, e.g., sarcoma, neuroblastoma, breast carcinoma among many others, c-Src has been found to be activated, particularly in colon cancers, especially in those metastatic to the liver (Rosen, N. et al. Analysis of pp60c-Src protein kinase activity in human tumor cell lines and tissues. J Biol Chem 261, 13754-13759 (1986); Bolen, J., Veillette, A., Schwartz, A., DeSeau, V. & Rosen, N. Activation of pp60c-Src protein kinase activity in human colon carcinoma. Proc. Natl. Acad. Sci. USA 84, 2251-2255 (1987); Cartwright, C., Kamps, M., Meisler, A., Pipas, J. & Eckhart, W. pp60c-Src activation in human colon carcinoma. J. Clin. Invest. 83, 2025-2033 (1989); Talamonti, M. A., Roh, M. S., Curley, S. A. & Gallick, G. E. Increase in activity and level of pp60c-Src in progressive stages of human colorectal cancer. J. Clin. Invest. 91, 53-60 (1991); Cartwright, C., Coad, C. & Egbert, B. Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis. J. Clin. Invest. 93, 509-515 (1994); Termuhlen, P. M., Curley, S. A., Talamonti, M. S., Saboorian, M. H. & Gallick, G. E. Site-specific differences in pp60c-Src activity in human colorectal metastases. J. Surg. Res. 54, 293-298 (1993); Mao, W. et al. Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential. Oncogene 15, 3083-3090 (1997)).
Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity can be downregulated by phosphorylation of an amino acid tyrosine at position 530 (Tyr 530 in human c-Src, which is equivalent to Tyr 527 in chicken Src) of the C-terminal regulatory region (Cooper, J., Gould, K., Cartwright, C. & Hunter, T. Tyr 527 is phosphorylated in pp60c-Src: implications for regulation. Science 231, 1431-1434 (1986); Cartwright, C., Eckhart, W., Simon, S. & Kaplan, P. Cell transformation by pp60c-Src mutated in the carboxy-terminal regulatory domain. Cell 49, 83-91 (1987); Kmiecik, T. & Shalloway, D. Activation and suppression of pp60c-Src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 49, 65-73 (1987); Piwnica-Worms, H., Saunders, K. B., Roberts, T. M., Smith, A. E. & Cheng, S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-Src. Cell 49, 75-82 (1987); Reynolds, A. B. et al. Activation of the oncogenic potential of the avian cellular Src protein by specific structural alteration of the carboxy terminus. Embo J. 6, 2359-2364 (1987); Jove, R., Hanafusa, T., Hamaguchi, M. & Hanafusa, H. In vivo phosphorylation states and kinase activities of transforming p60c-Src mutants. Oncogene Res. 5, 49-60 (1989); Bjorge, J. et al. Characterization of two activated mutants of human pp60c-Src that escape c-Src kinase regulation by distinct mechanisms. J. Biol. Chem. 270, 24222-24228 (1995)). It is possible that other mutations and phosphorylation processes involving tyrosine and other amino acids encoded by Src oncogene might be linked to tumorigenesis. For example, in chickens a single point mutation at residues Thr 338, Glu 378, Ile 441 or Arg 95 appears to activate the transforming ability of pp60c-Src (Wang P, Fromowitz F, Koslow M, Hagag N, Johnson B, Viola M. c-Src structure in human cancers with elevated pp60c-Src activity. Br J Cancer Sep; 64(3):531-3, 1991). However, according to the current state of the art, nothing has been identified in the human species that is as important as phosphorylation of Tyr 530 residue. For example, phosphorylation of Tyr 419 is not essential for tumor transformation (Snyder, M. A., Bishop, J. M., Colby, W. W. & Levinson, A. D. Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-Src. Cell 32, 891-901 (1983)). While this Tyr 530 mutation might be responsible for tumor formation it may not be the only cause and there is thus a continuing need to identify and further characterize the c-Src gene and pp60 as targets for drug discovery. The present inventors have surprisingly discovered for the first time that a novel mutation at SRC 531 is responsible for malignant transformation and metastasis. The existence of a mutant form of c-Src is disclosed that plays a role in Src activation in cancer.